HAART therapy has provided a significant benefit to the treatment of AIDS. Therapy fails in some patients, however, suggesting that better therapies are needed. We have shown in SIV-infected macaque that a combined regimen of DNA immunization and ART significantly reduced virus burden over than achieved by ART alone. However, similar to humans, some animals maintained a high virus burden despite treatment. We propose to use the monkey model to determine why this dichotomy exists so that therapy for humans nonresponsive to HAART can be developed. In project 2 we seek to address the hypothesis that the tissue reservoir for virus resides in the gut associated tissues (GALT). Further, individuals who maintain high viral loads during HAART do so because virus-specific immunity in the mucosa is either of insufficient magnitude (project 1) or dysfunctional (project 3). Thus, immunization with a DNA vaccine that induces mucosal responses will reduce virus burden. Two therapies will be evaluated: 1) a regimen mimicking the GlaxoSmithKline human DNA vaccine vector encoding gaglpo/nef that will be tested for immunogenicity in humans in 2003 and 2) the same DNA vaccine adjuvanted by codelivery of a vector encoding the potent mucosal adjuvant, E. coli heat-labile enterotoxin. We will perform a detailed comparison of virus burden, quasispecies distribution, and drug phenotype in the GALT vs. the peripheral circulation in animals enrolled in these studies. The responder/nonresponder phenotype of monkeys will be determined by quantification of plasma RNA burden by real time PCR and CD4+ T cells by flow cytometry. The role of the gut as the viral reservoir will be determined by quantifying cell associated viral DNA and RNA to determine the transcription rate in blood (PBMC, peripheral lymph nodes) and the GALT (jejunal lamina propria, mesenteric lymph nodes) using real time PCR. The quasispecies distribution of virus expressed at these sites will be determined by homoduplex mobility tracking assays (HTA) and confirmed by phylogenetic analysis. The selection/amplification of drug-resistant variants during HAART will also be revealed by testing virus in the periphery for in vitro drug resistance and identifying the in vivo tissue source by HTA. Together with analysis of the mucosal immune responses (projects 1 and 3), the impact of vaccines targeted to the mucosa will be identified.